Sequence-specific binding of oligonucleotides both to single-stranded RNA and DNA and to duplex DNA has been demonstrated. The appropriate sequence recognition for binding to single-stranded targets is well known: the A-T and G-C pairing characteristic of duplex formation has been established as the basis for DNA replication and transcription.
More recently, oligonucleotides have been shown to bind in a sequence-specific manner to duplex DNA to form triplexes. Single-stranded nucleic acid, primarily RNA, is the target molecule for oligonucleotides that are used to inhibit gene expression by an "antisense" mechanism (Uhlmann, E., et al., Chem Reviews (1990) 90:543-584; van der Krol, A. R., et al., Biotechniques (1988) 6:958-976). Antisense oligonucleotides are postulated to exert an effect on target gene expression by hybridizing with a complementary RNA sequence. In this model, the hybrid RNA-oligonucleotide duplex interferes with one or more aspects of RNA metabolism including processing, translation and metabolic turnover. Chemically modified oligonucleotides have been used to enhance their nuclease stability.
Duplex DNA can be specifically recognized by oligomers based on a recognizable nucleomonomer sequence. The motif termed "GT" recognition has been described (Beal, P. A., et al., Science (1992) 251:1360-1363; Cooney, M., et al., Science (1988) 241:456-459; Hogan, M. E., et al., EP Publication 375408). In the G-T motif, the oligonucleotide is oriented antiparallel to the target purine-rich sequence and A-T pairs are recognized by adenine or thymine residues and G-C pairs by guanine residues.
Sequence-specific targeting of both single-stranded and duplex target sequences has applications in diagnosis, analysis, and therapy. Under some circumstances wherein such binding is to be effected, it is advantageous to stabilize the resulting duplex or triplex over long time periods.
Covalent crosslinking of the oligomer to the target provides one approach to prolong stabilization. Sequence-specific recognition of single-stranded DNA accompanied by covalent crosslinking has been reported by several groups. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleomonomers complementary to target sequences. A report of similar work by the same group is that by Knorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleomonomer which was capable of activating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B., et al., J Am Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to a target nucleomonomer using an alkylating agent complementary to the single-stranded target nucleomonomer sequence. Photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Horne, et al., J Am Chem Soc (1990) 112:2435-2437.
Use of N.sup.4,N.sup.4 -ethanocytosine as an alkylating agent to crosslink to single-stranded and double-stranded oligomers has also been described (Webb and Matteucci, J. Am Chem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Shaw, J. P., et al., J Am Chem Soc (1991) 113:7765-7766). These papers also describe the synthesis of oligonucleotides containing the derivatized cytosine. Matteucci and Webb, in a later article in Tetrahedron Letters (1987) 28:2469-2472, describe the synthesis of oligomers containing N.sup.6,N.sup.6 -ethanoadenine and the crosslinking properties of this residue in the context of an oligonucleotide binding to a single-stranded DNA. Praseuth, D., et al., Proc Natl Acad Sci (USA) (1988) 85:1349-1353, described sequence-specific binding of an octathymidylate conjugated to a photoactivatable crosslinking agent to both single-stranded and double-stranded DNA.
In addition, Vlassov, V. V. et al., Gene (1988) 313-322 and Fedorova, O. S. et al., FEBS (1988) 228:273-276, describe targeting duplex DNA with an alkylating agent linked through a 5'-phosphate of an oligonucleotide.
In effecting binding to obtain a triplex, to provide for instances wherein purine residues are concentrated on one chain of the target and then on the opposite chain, oligomers of inverted polarity can be provided. By "inverted polarity" is meant that the oligomer contains tandem sequences which have opposite polarity, i.e., one segment or region of sequences having polarity 5'.fwdarw.3' followed by another with polarity 3'.fwdarw.5', or vice versa. This implies that these sequences are joined by linkages which can be thought of as effectively a 3'-3' internucleoside junction (however the linkage is accomplished), or effectively a 5'-5' internucleoside junction. Such oligomers have been suggested as by-products of reactions to obtain cyclic oligonucleotides by Capobionco, M. L., et al., Nucleic Acids Res (1990) 18:2661-2669. Compositions of "parallel-stranded DNA" designed to form hairpins secured with AT linkages using either a 3'-3' inversion or a 5'-5' inversion have been synthesized by van de Sande, J. H., et al., Science (1988) 241:551-557. In addition, triple helix formation using oligomers which contain 3'-3' linkages have been described (Horne, D. A., and Dervan, P. B., J Am Chem Soc (1990) 112:2435-2437; Froehler, B. C., et al., Biochemistry (1992) 31:1603-1609).
The use of triple helix (or triplex) complexes as a means for inhibition of the expression of target gene expression has been previously adduced (International Application No. PCT/US89/05769). Triple helix structures have been shown to interfere with target gene expression (International Application No. PCT/US91/09321; Young, S. L. et al., Proc Natl Acad Sci (1991) 88:10023-10026), demonstrating the feasibility of this approach.
Oligomers containing 5-propynyl modified pyrimidines have been described (Froehler, B. C., et al., Tetrahedron Letters (1992) 33:5307-5310; and Froehler, B. C., et al., Tetrahedron Letters (1993) 34:1003-1006).
2'-deoxy-7-deazaadenosine and 2'-deoxy-7-deazaguanosine have been incorporated into oligodeoxynucleotides and assessed for binding to the complementary DNA sequences. Thermal denaturation analysis (Tm) has shown that these substitutions modestly decrease the Tm of the duplex when these analogs are substituted for 2'-deoxyadenosine and 2'-deoxyguanosine (Seela, F. and Kehne, A., Biochemistry (1987) 26:2232-2238; and Seela, F. and Driller, H., Nucleic Acids Res (1986) 14:2319-2332). It has also been shown that oligonucleotides which alternate 2'-deoxy-7-deazaadenosine and thymidine can have a slightly enhanced duplex Tm over oligonucleotides containing 2'-deoxyadenosine and thymidine (Seela, F. and Kehne, A., Biochemistry (1985) 24:7556-7561).
2',3'-dideoxydeazapurine nucleosides have been used as chain terminators for DNA sequencing. 7-propargyl amino linkers are used for incorporation of fluorescent dyes into the nucleoside triphosphates (Prober, J. M. et al., Science (1987) 238:336-341).
DNA synthesis via amidite and hydrogen phosphonate chemistries has been described (U.S. Pat. Nos. 4,725,677; 4,415,732; 4,458,066; and 4,959,463).
Oligomers having enhanced affinity for complementary target nucleic acid sequences or enhanced nuclease stability would have improved properties for diagnostic applications, therapeutic applications and research reagents. Thus, a need exists for oligomers with one or both of these properties. Oligomers of the present invention have improved binding affinity for double stranded and/or single stranded target sequences or enhanced nuclease stability.